Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC) are severe and relapsing immunologically mediated chronic disorders of the gastrointestinal tract. IBDs are heterogeneous diseases characterized by various genetic abnormalities that lead to overly aggressive inflammatory responses to a subset of commensal enteric bacteria.
Crohn's disease can affect all parts of the digestive tract and specially the ileum and/or colon and leads to mucosal ulcerations, fistula, and deep infiltration of inflammatory cells in the bowel wall.
Ulcerative colitis often involves the lower part of the colon and the rectum and mucosal inflammation may extend to the caecum in a contiguous pattern.
The diagnosis of inflammatory bowel disease (IBD) is often achieved only months or years after the onset of symptoms. Several serological indicators of IBD have been identified; in general, they are antibodies directed against antigens expressed by organisms of the intestinal microbiome. For example, the anti-Saccharomyces cerevisiae antibody (ASCA) interacts with mannose epitopes of this yeast species and is present in 48% to 80% of patients with CD (Barahona-Garrido et al., (2009) Serological markers in inflammatory bowel disease: a review of their clinical utility. Rev Gastroenterol Mex.74(3):230-7). In general, these markers are specific for IBD, but experience low sensitivity. IBD biomarkers can also be of value after the diagnosis is established, as measures of disease activity and predictors of outcome. So far, endoscopy used in the diagnosis of IBD can be accurate but is invasive and expensive. Hence, there is still a need for biological markers of disease activity, which are simple to use in clinical practice, reliable and inexpensive.
Other serum and stool markers, such as C-reactive protein (CRP) and fecal calprotectin, are elevated in inflammatory and gastrointestinal diseases, but are not all specific for IBD (Desai, Fabion and Sandborn (2007) Review article: biological activity markers in inflammatory bowel disease. Aliment Pharmacol Ther 25, 247-255). The introduction of additional sensitive, specific, and noninvasive diagnostic markers may aid in the diagnosis of IBD, reduce patient risk and discomfort by reducing invasive testing, and accelerate the study of new treatments. One possibility is that miRNAs may serve as biomarkers for IBD.
MicroRNAs (miRNAs) are small (˜22-24 nucleotide), noncoding RNAs that act as key regulators of gene expression (Guarnieri D J, DiLeone R J. MicroRNAs: a new class of gene regulators. Ann Med. 2008; 40:197-208). Briefly, they are initially transcribed as longer primary miRNA transcripts in the nucleus then subsequently processed by Drosha and DGCR8 into precursor miRNA (pre-miRNA). The pre-miRNA is exported to the cytoplasm by exportin 5 in a ras-related nuclear protein guanosine triphosphate-dependent manner. The cytoplasmic pre-miRNA is cleaved by Dicer and the functional miRNA strand incorporated into the RNA-inducing silencing complex (RISC). Once loaded, the miRNA binds to complementary sequences in the 3′-untranslated region (3′UTR) of target miRNAs, resulting in suppression of translation and/or degradation of mRNA.
Overall, miRNAs are thought to contribute to the regulation of over 60% of all protein coding genes (Lewis Burge, Bartel (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell:120:15-20) including those involved in development, metabolism, cell cycle control and fibrosis (Guarnieri D J, DiLeone; Jiang X, Tsitsiou E, Herrick S E, et al. (2010) MicroRNAs and the regulation of fibrosis. FEBS J:277:2015).
It has been hypothesized that the differential expression of miRNAs may distinguish disease states (Perron M P, Boissonneault V, Gobeil L A, et al. (2006) Regulatory RNAs: Future Perspectives in Diagnosis, Prognosis, and Individualized Therapy. Methods Mol Biol:361:311-326). Indeed, altered miRNA profiles have been noted a vast array of diseases including multiple cancer subtypes (Cho W C. (2010) MicroRNAs in cancer—from research to therapy. Biochim Biophys Acta:1805:209-217) cardiovascular diseases (Catalucci D, Gallo P, Condorelli G. (2009) MicroRNAs in cardiovascular biology and heart disease. Circ Cardiovasc Genet:2:402-408), diabetes (Tang X, Tang G, Ozcan S. (2008) Role of microRNAs in diabetes. Biochim Biophys Acta:1779:697-701), and several inflammatory and autoimmune diseases (Sonkoly E, Wei T, Janson P C, et al. (2007) MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis? PLoS One:2:e610; Stanczyk J, Pedrioli D M, Brentano F, et al. (2008) Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis Rheum:58:1001-1009; Nakasa T, Miyaki S, Okubo A, et al. (2008) Expression of microRNA-146 in rheumatoid arthritis synovial tissue. Arthritis Rheum:58:1284-1292; Tan Z, Randall G, Fan J, et al. (2007) Allele-specific targeting of microRNAs to HLA-G and risk of asthma. Am J Hum Genet:81:829-834), including CD and UC (Wu et al., (2011) Peripheral blood microRNAs distinguish active ulcerative colitis and Crohn's disease. Inflamm Bowel Dis. 17(1): 241-250).
Wu et al profiled miRNA expression in blood of ulcerative colitis (UC) and Crohn's disease (CD) patients, namely active CD, inactive CD, active UC, inactive UC. Specifically, the blood expression of miRs-199a-5p, -362-3p, -340*, -532-3p and miRplus-1271 were elevated in both CD and UC as compared to healthy controls. In addition, miRs-28-5p, -151-5p, -199a-5p, -340* and miRplus-E1271 were increased in the peripheral blood of patients with active UC but not in inactive UC and miRs-199a-5p, -362-3p and -532-3p and miRplus-E1271 were increased in the peripheral blood of patients with active CD but not in the blood of patients with inactive CD as compared to healthy controls.
Fasseu et al., describes dysregulated miRNAs in colonic pinch biopsies in CD and UC (Fasseu et al., (2010) Identification of Restricted Subsets of Mature Abnormally Expressed in Inactive Colonic Mucosa Patients with Inflammatory Bowel Disease. Volume 5, Issue 10, e13160).
Similarly, Coskun et al. summarizes dysregulated miRNAs in IBD (Coskun et al., (2012) MicroRNAs in inflammatory bowel disease—pathogenesis, diagnostics and therapeutics. World J Gastroenterol; 18(34): 4629-4634). Also WO 2009/120877 describes differentaly regulated miRNAs in CD and UC from blood and biopsis.
An object of the present was therefore to provide an alternative method for detecting UC and CD.
The technical problem is solved by the embodiments reflected in the claims, described in the description, and illustrated in the Examples and Figures.
The above being said, the present invention relates to an in vitro method for diagnosing an acute or relapsing phase of inflammatory bowel disease in a subject, the method comprising                i) determining the level of a nucleic acid molecule comprising any one of SEQ ID NO: 1 or 2 in a test sample;        ii) comparing the level of the nucleic acid molecule determined in the test sample with a control sample;                    wherein an elevation of said nucleic acid molecule in the test sample is indicative of an acute or relapsing phase of said inflammatory bowel disease.                        
In one embodiment of the method of the present invention, the nucleic acid molecule has the SEQ ID NO: 1.
In another embodiment of the method of the present invention, the subject is a mammal, preferably a human being.
In another embodiment of the method of the present invention, the level of the S100A8 mRNA in said subject exhibits no alteration when compared to the control subject.
In another embodiment of the method of the present invention, the method further comprises the step of determining the S100A8 mRNA level in said test sample and optionally comparing it to the level in the control sample.
In another embodiment of the method of the present invention, the test sample and/or said control sample is any sample obtained not using endoscopy.
In another embodiment of the method of the present invention, the test sample and/or said control sample is selected from stool, urine, blood, salvia, preferably the test and/or control sample is a blood sample.
In another embodiment of the method of the present invention, the level of said nucleic acid molecule is determined by qRT-PCR.
In another embodiment of the method of the present invention, the level of S100A8 is determined by qRT-PCR.
In another embodiment of the method of the present invention, the method comprises the steps of                a) extracting RNA from the test sample;        b) performing qRT-PCR with specific oligonucleotides suitable to detect said nucleic acid molecule.        
In another embodiment of the method of the present invention, the level of the nucleic acid molecule is detected by a method comprising:                a) extracting RNA from said test sample;        b) treating the obtained RNA with a reverse transcription reaction mixture comprising specific oligonucleotides corresponding to the nucleic acid molecule of SEQ ID NO: 1 or 2, dNTPs and a reverse transcriptase under conditions allowing transcription of the nucleic acid molecule into complementary DNA (cDNA);        c) quantitative detection of cDNA transcripts of said nucleic acid molecule, wherein steps b) and c) can be either performed in separate reactions or in one reaction.        
The present invention further relates to a method for diagnosing the acute or relapsing phase of inflammatory bowel disease comprising:                (a) determining the level of SEQ ID NO: 1 or 2 in a test sample;        (b) determining the level of S100A8 mRNA in said test sample; and        (c) comparing both levels to a control sample or control value;        wherein an increase of said SEQ ID and no alteration of said S100A8 in comparison to said control sample or control value indicates the acute or relapsing phase of inflammatory bowel disease.        
The present invention also relates to a method for treating the acute or relapsing phase of inflammatory bowel disease in a subject comprising:                (a) determining the level of any of SEQ ID NO: 1 or 2 in a test sample;        (b) determining the level of S100A8 mRNA in said test sample;        (c) comparing both levels to a control sample or control value; and        (d) treating said subject with MEDICAMENT, provided that said test sample is characterized by an increase of said SEQ ID and by no alteration of said S100A8 in comparison to said control sample or control value.        
Additionally, the present invention relates to a MEDICAMENT for use in the treatment of inflammatory bowel disease in a subject, wherein said subject is characterized by an increase of SEQ ID NO: 1 or 2 and by no alteration of S100A8 mRNA in comparison to a control subject or control value.
The present invention also relates to the use of the level of SEQ ID NO: 1 or 2 in a test sample of a subject suffering from inflammatory bowel disease, for monitoring the progression of said disease in said subject.
Further, the present invention relates to a use of a nucleic acid molecule selected of any of SEQ ID NO: 1 or 2 for in vitro diagnosis of an acute or relapsing phase of inflammatory bowel disease.
In one embodiment, the use of the nucleic acid molecule selected of any of SEQ ID NO: 1 or 2 for in vitro diagnosis includes a subject that has an unchanged expression of S100A8 mRNA as compared to a control subject, preferably the control subject is a healthy subject.
The present invention also relates to a device for the diagnosis of an acute or relapsing phase of inflammatory bowel disease of a subject, wherein the device comprises oligonucleotide sequences to which any of SEQ ID NO: 1 or 2 hybridizes to detect the level of a nucleic acid molecule of SEQ ID NO: 1 or 2 in a test sample.
Additionally, the present invention relates to a kit comprising one or more extraction buffer/reagents and protocol; reverse transcription buffer/reagents and protocol; and qPCR buffer/reagents and protocol suitable for performing any of the methods of the present invention.